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• W1988811776 abstract "Protein kinase C θ (PKCθ) is a novel Ca2+-independent PKC isoform, which is selectively expressed in skeletal muscle and hematopoietic cells, especially T cells. In T cells, it colocalizes with the T cell antigen receptor (TCR)·CD3 complex in antigen-stimulated T cells and is involved in the transcriptional activation of the interleukin-2 gene. In the present study, we report that PKCθ is tyrosine phosphorylated in Jurkat T cells upon TCR·CD3 activation. The Src family protein-tyrosine kinase, Lck, was critical in TCR-induced tyrosine phosphorylation of PKCθ. Lck phosphorylated and was associated with the regulatory domain of PKCθ both in vitro and in intact cells. This association was constitutive, but it was enhanced by T cell activation, with both Src-homology 2 and Src-homology 3 domains of Lck contributing to it. Tyrosine 90 (Tyr-90) in the regulatory domain of PKCθ was identified as the major phosphorylation site by Lck. A constitutively active mutant of PKCθ (A148E) could enhance proliferation of Jurkat T cells and synergized with ionomycin to induce nuclear factor of T cells activity. However, mutation of Tyr-90 into phenylalanine markedly reduced (or abolished) these activities. These results suggest that Lck plays an important role in tyrosine phosphorylation of PKCθ, which may in turn modulate the physiological functions of PKCθ during TCR-induced T cell activation. Protein kinase C θ (PKCθ) is a novel Ca2+-independent PKC isoform, which is selectively expressed in skeletal muscle and hematopoietic cells, especially T cells. In T cells, it colocalizes with the T cell antigen receptor (TCR)·CD3 complex in antigen-stimulated T cells and is involved in the transcriptional activation of the interleukin-2 gene. In the present study, we report that PKCθ is tyrosine phosphorylated in Jurkat T cells upon TCR·CD3 activation. The Src family protein-tyrosine kinase, Lck, was critical in TCR-induced tyrosine phosphorylation of PKCθ. Lck phosphorylated and was associated with the regulatory domain of PKCθ both in vitro and in intact cells. This association was constitutive, but it was enhanced by T cell activation, with both Src-homology 2 and Src-homology 3 domains of Lck contributing to it. Tyrosine 90 (Tyr-90) in the regulatory domain of PKCθ was identified as the major phosphorylation site by Lck. A constitutively active mutant of PKCθ (A148E) could enhance proliferation of Jurkat T cells and synergized with ionomycin to induce nuclear factor of T cells activity. However, mutation of Tyr-90 into phenylalanine markedly reduced (or abolished) these activities. These results suggest that Lck plays an important role in tyrosine phosphorylation of PKCθ, which may in turn modulate the physiological functions of PKCθ during TCR-induced T cell activation. protein kinase C T cell antigen receptor protein-tyrosine kinase monoclonal antibodies phosphotyrosine glutathioneS-transferase Src-homology regulatory domain nuclear factor of T cells catalytic domain Protein kinase C (PKC)1is a family of serine/threonine kinases that play critical roles in the regulation of differentiation and proliferation in many cell types and in the response to diverse stimuli (reviewed in Refs. 1.Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2347) Google Scholar and 2.Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (843) Google Scholar). Products of the 10 known mammalian PKC genes are classified into four subfamilies of Ca2+-dependent (or conventional, PKCα, -β, and -γ), Ca2+-independent (or novel, PKCδ, -ε, -η, and -θ), atypical (PKCζ and ι/λ), and PKCμ/D enzymes. Activity of PKC enzymes is regulated by phosphorylation and binding of defined cofactors. Enzyme activation is associated with its redistribution among different cellular compartments, commonly from the cytosolic to the particulate (membrane) fraction. Studies indicate that PKC is also important during T cell activation. This is indicated by the ability of physiological T cell receptor (TCR) ligands to activate PKC and induce its translocation from the cytosol to the particulate fraction; by the ability of PKC inhibitors, or PKC depletion by prolonged phorbol ester treatment, to block lymphocyte signaling and activation; by the requirement for persistent PKC activation during mitogenic T cell activation; and, finally, by the diminished TCR·CD3-mediated proliferation in PKC-deficient T cells (reviewed in Ref. 3.Altman A. Coggeshall K.M. Mustelin T. Adv. Immunol. 1990; 48: 227-360Crossref PubMed Google Scholar). PKCθ is a novel Ca2+-independent PKC isoform. It is characterized by a unique tissue distribution, i.e. in skeletal muscle, lymphoid organs, and hematopoietic cell lines, in particular T cells (4.Osada S. Mizuno K. Saido T.C. Suzuki K. Kuroki T. Ohno S. Mol. Cell. Biol. 1992; 12: 3930-3938Crossref PubMed Google Scholar, 5.Baier G. Telford D. Giampa L. Coggeshall K.M. Baier-Bitterlich G. Isakov N. Altman A. J. Biol. Chem. 1993; 268: 4997-5004Abstract Full Text PDF PubMed Google Scholar, 6.Chang J.D. Xu Y. Raychowdhury M.K. Ware J.A. J. Biol. Chem. 1993; 268: 14208-14214Abstract Full Text PDF PubMed Google Scholar); by isoenzyme-specific activation requirements and substrate preferences in vitro (7.Baier G. Baier-Bitterlich G. Meller N. Coggeshall K.M. Telford D. Giampa L. Isakov N. Altman A. Eur. J. Biochem. 1994; 225: 195-203Crossref PubMed Scopus (78) Google Scholar, 8.Pietromonaco S.F. Simons P.C. Altman A. Elias L. J. Biol. Chem. 1998; 273: 7594-7603Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar); and by its presence in the particulate and detergent-insoluble (i.e. cytoskeletal) fraction in resting T cells (unlike,e.g. PKCα and -β) (9.Meller N. Liu Y.C. Collins T.L. Bonnefoy-Bérard N. Baier G. Isakov N. Altman A. Mol. Cell. Biol. 1996; 16: 5782-5791Crossref PubMed Google Scholar). Previous reports have shown that among several PKC isoforms tested, only PKCθ was capable of significantly stimulating Ras-dependent transcription from an AP-1 element in EL4 leukemic T cells (10.Baier-Bitterlich G. Übberall F. Bauer B. Fresser F. Wachter H. Grünicke H. Utermann G. Altman A. Baier G. Mol. Cell. Biol. 1996; 16: 1842-1850Crossref PubMed Google Scholar). PKCθ also specifically cooperates with calcineurin and plays a critical role in c-Jun NH2-terminal kinase activation and induction of the interleukin-2 gene (11.Werlen G. Jacinto E. Xia Y. Karin M. EMBO J. 1998; 17: 3101-3111Crossref PubMed Scopus (252) Google Scholar, 12.Ghaffari-Tabrizi N. Bauer B. Altman A. Utermann G. Uberall F. Baier G. Eur. J. Immunol. 1999; 29: 132-142Crossref PubMed Scopus (108) Google Scholar). Recent reports indicated that among different T cell-expressed PKC isoforms, PKCθ was the only one to colocalize precisely with the TCR·CD3 complex in the contact region between antigen-specific T cells and antigen-presenting cells. Importantly, this colocalization occurred at a high stoichiometry and correlated with positive activation signals leading to proliferation, as opposed to activation conditions, which result in anergy or apoptosis (13.Monks C.R.F. Kupfer H. Tamir I. Barlow A. Kupfer A. Nature. 1997; 385: 83-86Crossref PubMed Scopus (489) Google Scholar, 14.Monks C.R. Freiberg B.A. Kupfer H. Sciaky N. Kupfer A. Nature. 1998; 395: 82-86Crossref PubMed Scopus (1945) Google Scholar). These findings strongly suggest that PKCθ plays specialized role(s) in T cells as a specific constituent of signaling cascades that are involved in TCR·CD3-mediated T cell activation. One of the earliest signaling events in T cell activation via the TCR·CD3 complex is the activation of the Src and Syk families of protein-tyrosine kinases (PTKs), which in turn leads to the phosphorylation of numerous cellular proteins (15.Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1941) Google Scholar). There is evidence that cross-talk among PTKs and Ser/Thr kinases occurs commonly in different cell types and serves as an important regulatory mechanism. In this regard, recent studies have shown that a novel PKC, PKCδ, can be phosphorylated on tyrosine residues upon activation (16.Li W., Yu, J.C. Michieli P. Beeler J.F. Ellmore N. Heidaran M.A. Pierce J.H. Mol. Cell. Biol. 1994; 14: 6727-6735Crossref PubMed Google Scholar). Because of the close structural relationship between PKCδ and PKCθ (5.Baier G. Telford D. Giampa L. Coggeshall K.M. Baier-Bitterlich G. Isakov N. Altman A. J. Biol. Chem. 1993; 268: 4997-5004Abstract Full Text PDF PubMed Google Scholar) and the finding that PKCθ colocalizes to the activated TCR complex (which includes activated PTKs) in antigen-specific T cells (13.Monks C.R.F. Kupfer H. Tamir I. Barlow A. Kupfer A. Nature. 1997; 385: 83-86Crossref PubMed Scopus (489) Google Scholar), we have decided to examine whether PKCθ can become phosphorylated on tyrosine residues. Anti-Lck and -glutathioneS-transferase (GST) monoclonal antibodies (mAbs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-phosphotyrosine Tyr(P) mAb (4G10) was purchased from Upstate Biotechnology (Lake Placid, NY), and the anti-PKCθ mAb was from Transduction Laboratories (Lexington, KY). The anti-human CD3 mAb, OKT3, was purified from culture supernatants of the corresponding hybridoma by protein A-Sepharose chromatography. The anti-hemagglutinin (clone 12CA5) and -Xpress™ tag mAbs were obtained from Roche Molecular Biochemicals and Invitrogen (Carlsbad, CA), respectively. A goat anti-mouse Ig antibody was obtained from Pierce. GST fusion proteins containing the amino-terminal SH2 and SH3 or the combined amino-terminal, SH2, and SH3 domains of Lck (GST-Lck/N, GST-Lck/SH2, GST-Lck/SH3, and GST-Lck/N+3+2, respectively) were generated as described previously (17.Amrein K.E. Panholzer B. Flint N.A. Bannwarth W. Burn P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10285-10289Crossref PubMed Scopus (37) Google Scholar). Synthetic 15-mer peptides containing the various tyrosine residues present in the regulatory domain of PKCθ (Fig. 5 B) were from QCB, Hopkinton, MA. Simian virus 40 large T antigen-transfected human leukemic Jurkat T cells (Jurkat-TAg), wild-type Jurkat cells, and Lck-deficient (J.CaM1.6 (18.Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (920) Google Scholar)) or ZAP-70-deficient (P116 (19.Williams B.L. Schreiber K.L. Zhang W. Wange R.L. Samelson L.E. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1998; 18: 1388-1399Crossref PubMed Scopus (222) Google Scholar)) variant Jurkat cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mml-glutamine, 1 mmsodium pyruvate, 100 μm minimal Eagle's medium nonessential amino acids, 10 mm HEPES, and 50 μm β-mercaptoethanol and antibiotics. In some experiments, the cells were stimulated for the indicated time periods with OKT3 (2 μg/ml) followed by cross-linking with a secondary goat anti-mouse Ig antibody, or with sodium orthovanadate (100 μm). COS-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 1 mm sodium pyruvate, and antibiotics. Full-length wild-type, constitutively active (A148E) or dominant-negative (K409R) human PKCθ (10.Baier-Bitterlich G. Übberall F. Bauer B. Fresser F. Wachter H. Grünicke H. Utermann G. Altman A. Baier G. Mol. Cell. Biol. 1996; 16: 1842-1850Crossref PubMed Google Scholar) and Lck (20.Williams S. Couture C. Gilman J. Jascur T. Deckert M. Altman A. Mustelin T. Eur. J. Biochem. 1997; 245: 84-90Crossref PubMed Scopus (45) Google Scholar) cDNAs were generated as described previously. The PKCθ plasmids were subcloned into the BamHI andXbaI sites of the pEF4/His-C mammalian expression vector (Invitrogen) by standard techniques. This vector encodes in-frame 6xHis and XpressTM tags upstream of the insert. A constitutively active PKCθ-A148E plasmid in which Tyr-90 has been mutated to phenylalanine (Y90F) was made by site-directed mutagenesis. The cDNAs encoding the regulatory domain (PKCθ-RD, amino acid residues 1–378) or catalytic domain (PKCθ-CD, residues 379–706) of PKCθ were subcloned into a mammalian expression vector, pEFneo (21.Liu Y.C. Kawagishi M. Mikayama T. Inagaki Y. Takeuchi T. Ohashi H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8957-8961Crossref PubMed Scopus (43) Google Scholar), which has been tagged with an hemagglutinin epitope. A GST-PKCθ-RD fusion protein was prepared by cloning the corresponding cDNA into the pGEX-5X-1 Escherichia coli expression vector and purifying the expressed, isopropyl-1-thio-β-d-galactopyranoside-induced protein on glutathione-Sepharose beads (22.Bonnefoy-Bérard N. Liu Y.-C. von Willebrand M. Sung A. Elly C. Mustelin T. Yoshida H. Ishizaka K. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10142-10146Crossref PubMed Scopus (133) Google Scholar). The NFAT/AP-1 and AP-1-luciferase reporter constructs were provided by G. Crabtree and M. Karin, respectively. A chloramphenicol acetyltransferase reporter gene driven by five repeats of the NFAT site (κ3 element) derived from the tumor necrosis factor α promoter was obtained from A. Rao. Jurkat-TAg and COS-1 cells were transiently transfected with 5–10 μg of cDNA by electroporation (260 V, 950 microfarads). Cells were cultured for 48–60 h before they were used in various assays. Cells were lysed in lysis buffer containing 1% Nonidet P-40, 20 mm Tris-HCl (pH 7.4), 150 mmNaCl, 5 mm NaF, 5 mm NaPP, 1 mmNa3VO4, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml aprotinin and leupeptin. Cell lysates were mixed with antibody for 1 h at 4 °C and then incubated with 30 μl of protein G-Sepharose beads (Amersham Pharmacia Biotech) for an additional hour. Binding reactions containing 10 μg of GST fusion proteins and cell lysates were incubated for 2 h at 4 °C, followed by the addition of 20 μl of glutathione-Sepharose 4B beads and incubation for 1 h at 4 °C. Precipitates were washed five times with lysis buffer and boiled in 30 μl of sample buffer for 5 min. Samples were subjected to SDS-polyacrylamide gel electrophoresis analysis and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were immunoblotted with primary antibodies overnight at 4 °C or for 2 h at room temperature. After a brief wash, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The membranes were washed and visualized by the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech). COS-1 cells transiently transfected with Lck were lysed, and Lck was immunoprecipitated from 1 × 106 cells as described above. Immunoprecipitates were washed four times in lysis buffer and one time in 50 mm HEPES (pH 7.0). 100 μl of reaction mixture containing 50 mm HEPES (pH 7.0), 10 mm MgCl2, 10 mm MnCl2, 1 μm ATP, 10 μCi of [γ-32P]ATP, 0.1% Nonidet P-40, and 10 μg of peptide substrate were added to immunoprecipitates and incubated at 30 °C for 30 min. The reactions were terminated by placing the samples on ice. After a brief spin, 25 μl of the reaction supernatant were transferred to SpinZyme phosphocellulose units (Pierce) and washed as per the manufacturer's instructions. 32PO4incorporation was determined in a Beckman LS 6500 scintillation counter. Jurkat-TAg cells were transfected with 5 μg of the appropriate reporter plasmid together with 5 μg of the indicated expression plasmids. Identical amounts of the corresponding empty vectors were used as controls. Transfection efficiencies were monitored by cotransfection of a thymidine kinase promoter luciferase reporter and using the Dual Luciferase kit (Promega, Madison, WI) according to the manufacturer's instructions. Cells were cultured for 24 h and either left unstimulated or activated for the final 6 h of culture with the indicated stimuli. The cells were lysed, and luciferase activity was determined as described previously (23.Liu Y.-C. Elly C. Langdon W.Y. Altman A. J. Biol. Chem. 1997; 272: 168-173Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The results are expressed as normalized luciferase activity of triplicate samples. Jurkat-Tag cells were transfected with the appropriate plasmid and plated on 96-well plates (1 × 104 cells/well). Cells were cultured for 48 h, pulsed with 0.5 μCi of [3H]thymidine for the last 6 h, and then harvested. [3H]thymidine incorporation was determined in a Beckman LS 6500 scintillation counter. Recent studies have shown that activation of different receptors leads to tyrosine phosphorylation of PKCδ in various cell types (16.Li W., Yu, J.C. Michieli P. Beeler J.F. Ellmore N. Heidaran M.A. Pierce J.H. Mol. Cell. Biol. 1994; 14: 6727-6735Crossref PubMed Google Scholar, 24.Haleem-Smith H. Chang E.-Y. Szalassi Z. Blumberg P.M. Rivera J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9112-9116Crossref PubMed Scopus (69) Google Scholar, 25.Denning M.F. Dlugosz A.A. Threadgill D.W. Magnuson T. Yuspa S.H. J. Biol. Chem. 1996; 271: 5325-5331Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 26.Moussazadeh M. Haimovich B. FEBS Lett. 1998; 438: 225-230Crossref PubMed Scopus (28) Google Scholar, 27.Song J.S. Swann P.G. Szallasi Z. Blank U. Blumberg P.M. Rivera J. Oncogene. 1998; 16: 3357-3368Crossref PubMed Scopus (97) Google Scholar). Because of the similarity between PKCδ and PKCθ we have decided to examine whether PKCθ can also become phosphorylated on tyrosine. As shown in Fig.1 A, PKCθ from resting Jurkat T cells contained a low level of Tyr(P). Anti-CD3 or pervanadate stimulation caused a marked increase in this phosphorylation. Unlike PKCδ in a murine myeloid progenitor cell line 32D (16.Li W., Yu, J.C. Michieli P. Beeler J.F. Ellmore N. Heidaran M.A. Pierce J.H. Mol. Cell. Biol. 1994; 14: 6727-6735Crossref PubMed Google Scholar), PKCθ was not phosphorylated on tyrosine in Jurkat T cells when stimulated with phorbol ester. To determine the kinetics of OKT3-induced tyrosine phosphorylation of PKCθ, Jurkat T cells were either left unstimulated or treated with OKT3 for different times. As shown in Fig. 1 B, OKT3-induced tyrosine phosphorylation of PKCθ peaked at 1 min, decreased thereafter, and returned to the baseline level within 30 min. To determine which T cell-expressed PTKs mediate the phosphorylation of PKCθ, COS-1 cells were cotransfected with PKCθ plus Lck, Fyn, ZAP-70, Syk, Itk (Emt), or combinations of Lck plus ZAP-70 or Lck plus Itk. PKCθ was immunoprecipitated, and its tyrosine phosphorylation was analyzed by anti-Tyr(P) immunoblotting. All these tyrosine kinases, with the exception of Lck, did not induce detectable tyrosine phosphorylation of PKCθ (data not shown). As shown in Fig.1 C, PKCθ was phosphorylated on tyrosine residue(s) in cells coexpressing Lck. This phosphorylation was not present in PKCθ plus empty vector-cotransfected cells. Moreover, immunoprecipitated endogenous PKCθ was phosphorylated in vitro on tyrosine by purified Lck (Fig. 1 D). PKCθ expression in the immunoprecipitates was monitored by immunoblotting and was found to be equivalent among different groups in each experiment (Fig. 1,bottom panels). Several recent studies reported that PKCδ associates with Src family PTKs, and is phosphorylated on tyrosine, in transformed cells (28.Zang Q. Lu Z. Curto M. Barile N. Shalloway D. Foster D.A. J. Biol. Chem. 1997; 272: 13275-13280Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 29.Shanmugam M. Krett N.L. Peters C.A. Maizels E.T. Murad F.M. Kawakatsu H. Rosen S.T. Hunzicker-Dunn M. Oncogene. 1998; 16: 1649-1654Crossref PubMed Scopus (56) Google Scholar) and in activated mast cells (27.Song J.S. Swann P.G. Szallasi Z. Blank U. Blumberg P.M. Rivera J. Oncogene. 1998; 16: 3357-3368Crossref PubMed Scopus (97) Google Scholar). To investigate whether PKCθ associates with Lck, we first performed in vitro binding assays using GST-Lck fusion proteins. Whole cell lysates from unstimulated, OKT3-, or pervanadate-stimulated Jurkat cells were incubated with GST alone, GST-Lck/N+3+2 (amino acid residues 1–244), GST-Lck/SH2 (residues 120–226), GST-Lck/SH3 (residues 54–120), or GST-Lck/N (residues 1–95). The precipitates were then analyzed with an anti-PKCθ antibody. As shown in Fig. 2 A, endogenous PKCθ from unstimulated cells was precipitated by GST-Lck/N+2+3, GST-Lck/SH2, and GST-Lck/SH3 proteins, but not by GST or GST-Lck/N. The association between PKCθ and GST-Lck/N+2+3 was enhanced when the cells were stimulated with pervanadate, whereas the association between PKCθ and GST-Lck/SH3 or GST-Lck/SH2 was enhanced by either OKT3 or pervanadate stimulation. Conversely, a GST-PKCθ-RD fusion protein (amino acids 1–378) was capable of binding Lck present in T cell lysates (Fig. 2 B). To address the question whether Lck and PKCθ associate with each other in vivo, Jurkat T cells were cotransfected with Lck and PKCθ expression plasmids. Probing of Lck immunoprecipitates from these cells with an anti-PKCθ mAb revealed that PKCθ coimmunoprecipitated with Lck; however, the amount of PKCθ associated with Lck did not change following anti-CD3 stimulation (Fig.2 C). When the reverse experiments were conducted using the anti-PKCθ mAb for immunoprecipitation, no Lck could be detected in association with PKCθ. It is possible that our immunoprecipitating antibody, which binds to the amino-terminal region of PKCθ, disrupts the association between Lck and PKCθ, because we found that the regulatory domain of PKCθ is involved in the interaction with Lck (Fig. 2 B). Overlay binding experiments were next carried out to examine whether the association between PKCθ and Lck is direct. Endogenous PKCθ from Jurkat cells was immunoprecipitated and transferred onto nitrocellulose membranes. The membranes were incubated with GST-Lck fusion proteins, and membrane-bound fusion proteins were detected with an anti-GST mAb. As shown in Fig. 3, the SH3, SH2, and the full regulatory domain of Lck (Lck/N+3+2) bound directly to PKCθ. This binding was not significantly affected by stimulation, with the exception of pervanadate stimulation, which enhanced the binding of the Lck SH2 domain to the membrane-bound PKCθ. To address the role of Lck versus ZAP-70 in the phosphorylation of PKCθ, we compared the tyrosine phosphorylation of PKCθ in wild-type Jurkat T to that occurring in two mutant Jurkat cell lines, i.e. Lck-deficient (J.CaM1.6 (18.Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (920) Google Scholar)) or ZAP-70-deficient (P116 (19.Williams B.L. Schreiber K.L. Zhang W. Wange R.L. Samelson L.E. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1998; 18: 1388-1399Crossref PubMed Scopus (222) Google Scholar)) Jurkat T cells. As shown in Fig.4, the basal tyrosine phosphorylation of PKCθ was abrogated in the Lck-deficient cells, whereas the OKT3- or pervanadate-mediated tyrosine phosphorylation was greatly reduced in the same cells. In contrast, the basal as well as the prominent pervanadate-induced tyrosine phosphorylation of PKCθ was maintained in P116 cells, but its anti-CD3-induced phosphorylation was reduced to a level approaching the basal one. These data suggest that Lck plays a critical role in the tyrosine phosphorylation of PKCθ in T cells and that ZAP-70 may contribute to this event under basal conditions or in anti-CD3-stimulated cells, although it is largely dispensable for the pervanadate-induced phosphorylation of PKCθ. To map the site(s) in PKCθ phosphorylated by Lck, we first determined whether the RD, the CD, or both can be phosphorylated by Lck in transiently transfected cells. The regulatory domain of PKCθ was prominently phosphorylated on tyrosine in COS-1 cells coexpressing Lck (Fig. 5 A, left panels), whereas tyrosine phosphorylation of the catalytic domain was not detectable under the same conditions (Fig. 5 A,right panels). Next, a series of PKCθ-derived 15-mer peptides containing the various tyrosine residues present in the regulatory domain were used as substrates in in vitro Lck kinase assays. All of these peptides contained tyrosine in the center position, with the exception of the peptide representing Tyr-237 and Tyr-239 (Fig. 5 B). Only the peptide containing Tyr-90 was a good substrate for Lck isolated by immunoprecipitation from Lck-overexpressing COS-1 cells (Fig. 5 C). To determine whether Tyr-90, which is located in the V1 region within the regulatory domain of PKCθ, represents a substrate for Lck in intact cells, we replaced this tyrosine residue with phenylalanine by site-directed mutagenesis and then compared the tyrosine phosphorylation of this mutant versus wild-type PKCθ in transfected COS-1 or Jurkat cells. Both full-length PKCθ and its regulatory domain were assayed in these experiments. When cotransfected with Lck into COS-1 cells, the level of Tyr(P) in both the regulatory domain (Fig. 6 A, top left panel) and the full-length (Fig. 6 A, top right panel) Y90F-mutated proteins was markedly reduced (≥90%) in comparison with the corresponding wild-type PKCθ proteins, despite the similar expression levels of both PKCθ (middle panels) and Lck (bottom panels) in the two transfected groups. Tyrosine phosphorylation of PKCθ was not observed in cells transfected with empty vector instead of Lck (data not shown; Fig.1 C). Similarly, the anti-CD3- or pervanadate-induced tyrosine phsophorylation of the transfected PKCθ-RD in Jurkat T cells was greatly reduced when Tyr-90 was mutated to phenylalanine (Fig.6 B, top panel). All groups expressed similar levels of the transfected PKCθ-RD (bottom panel). Together, these experiments reveal that Tyr-90 in the regulatory domain of PKCθ is most likely the major phosphorylation site for Lck. To determine whether phosphorylation of Tyr-90 is important for the proper function of PKCθ in T cells, we compared two forms of constitutively active PKCθ (A148E), i.e. one containing the wild-type Tyr-90 and another in which the Y90F mutation has been introduced, in several functional assays. First, we evaluated the effect of mutating Tyr-90 on the in vitro catalytic activity of transfected PKCθ immunoprecipitated from transfected COS-1 cells (which do not express endogenous PKCθ), using myelin basic protein as a substrate. No significant differences were detected between wild-type and Y90F-mutated PKCθ either in the presence or absence of lipid cofactors (data not shown). Next, we evaluated the effect of the mutation on two downstream events that we recently found to be selectively induced by PKCθ, i.e. enhanced proliferation of Jurkat T cells; and second, we activated the NFAT-luciferase reporter gene in conjunction with a second signal provided by Ca2+ ionophore. As shown in Fig.7 A, the constitutively active PKCθ mutant containing Tyr-90 enhanced the proliferation of Jurkat cells by ∼50%. The actual level of enhancement is most likely considerably higher given the fact that only a relatively small fraction of the cells actually expresses the A148E mutant under these transient transfection conditions. Under the same conditions, the double PKCθ mutant (A148E/Y90F) was devoid of this activity. Similarly, the A148E/Y90F double mutant was deficient in inducing the activity of a reporter gene driven by an NFAT/AP-1 element derived from the interleukin-2 gene promoter (Fig. 7 B). These deficiencies did not reflect lower expression of the Y90F mutant, because immunoblotting with a tag-specific antibody indicated that the Y90F mutant was expressed as well as, or even better than, the single A148E mutant (Fig. 7, A and B, bottom panels). To further evaluate the specificity of this effect, as well as rule out the possibility that mutation of Tyr-90 nonselectively inactivates the enzyme or confers upon it a dominant-negative phenotype, we compared the ability of the same PKCθ mutants to activate reporter genes driven by isolated AP-1 or NFAT response elements. The results demonstrate that the double mutant was fully active in inducing AP-1 activity (Fig. 7 C) but was deficient relative to the single A/E mutant in stimulating NFAT activity (Fig. 7 D). These results indicate that Tyr-90 in PKCθ plays a role in NFAT, but not AP-1, activation and, furthermore, that the Tyr-90-mutated PKCθ does not behave in a nonselective inhibitory manner. Protein kinase C isozymes consist of an amino-terminal regulatory domain and a highly conserved carboxyl-terminal catalytic domain. The activation and localization of PKC enzymes are regulated by the binding of lipid cofactors and, in the case of conventional PKCs, also Ca2+, to the regulatory domain of PKC (2.Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (843) Google Scholar). PKC is also regulated by trans- and autophosphorylation on serine and threonine residues in the activation loop and the carboxyl-terminal region of the catalytic domain, modifications that are necessary for processing catalytically competent enzymes and for the correct subcellular localization of PKC (30.Newton A.C. Curr. Biol. 1995; 5: 973-976Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 31.Tsutakawa S.E. Medzihradszky K.F. Flint A.J. Burlingame A.L. Koshland Jr., D.E. J. Biol. Chem. 1995; 270: 26807-26812Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 32.Newton A.C. Johnson J.E. Biochim. Biophys. Acta. 1998; 1376: 155-172Crossref PubMed Scopus (242) Google Scholar, 33.Edwards A.S. Faux M.C. Scott J.D. Newton A.C. J. Biol. Chem. 1999; 274: 6461-6468Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). In a recent report, threonine 250 of PKCα has been identified as an autophosphorylation site upon 12-O-tetradecanoylphorbol-13-acetate stimulation (34.Ng T. Squire A. Hansra G. Bornancin F. Prevostel C. Hanby A. Harris W. Barnes D. Schmidt S. Mellor H. Bastiaens P.I. Parker P.J. Science. 1999; 283: 2085-2089Crossref PubMed Scopus (267) Google" @default.
• W1988811776 created "2016-06-24" @default.
• W1988811776 creator A5004326219 @default.
• W1988811776 creator A5023116987 @default.
• W1988811776 creator A5036104357 @default.
• W1988811776 creator A5041154564 @default.
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• W1988811776 date "2000-02-01" @default.
• W1988811776 modified "2023-10-16" @default.
• W1988811776 title "Regulation of Protein Kinase Cθ Function during T Cell Activation by Lck-mediated Tyrosine Phosphorylation" @default.
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